a-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in a-helix (the a domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in b sheet (the b domain, has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine a-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact a-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. The two disulfide form of a-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxylated cysteins, retains about half the secondary and tertiary structure of the intact a-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at low temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, a lactalbumin retains its calcium-binding b-domain, whereas the a-domain is unfolded. It appears that the b-domain does not require a-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded a-domain.